Polyhydroxyalkanoate synthase and gene encoding the same enzyme

ABSTRACT

A novel polyhydroxyalkanoate (PHA) synthase derived from a microorganism capable of producing a PHA having a novel side-chain structure and a DNA encoding the amino acid sequence for the synthase are provided. Two PHA synthase proteins (SEQ ID Nos. 1 and 3) derived from  Pseudomonas jessenii  P161 (FERM BP-7376) and PHA synthase genes encoding these PHA synthases are provided, respectively (SEQ ID Nos. 2 and 4). A recombinant microorganism is endowed with a PHA producing ability.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to a polyhydroxyalkanoate (hereinafter,referred to as a “PHA”) synthase, a gene encoding the PHA synthase, arecombinant vector containing the gene, a transformant capable ofexpressing the PHA synthase which has been transformed by therecombinant vector, a process for producing the PHA synthase utilizingthe transformant, and a process for preparing the PHA utilizing thetransformant. In particular, this invention relates to amicroorganism-derived PHA synthase capable of producing apolyhydroxyalkanoate and a gene encoding the PHA synthase utilized forexpressing the PHA synthase by transformation.

[0003] 2. Related Background Art

[0004] There have been reported a number of microorganisms producingpoly-3-hydroxybutyric acid (PHB) or another PHA and storing it therein(“Biodegradable Plastic Handbook”, edited by Biodegradative PlasticResearch Society, NTS Co. Ltd., p.178-197). These polymers may be, asconventional plastics, used for producing a variety of products by, forexample, melt-processing. Since they are biodegradable, they have anadvantage that they can be completely degraded by microorganisms in thenatural environment, and they do not cause pollution due to remaining inthe natural environment like many conventional polymer compounds.Furthermore, they are excellently biocompatible, and thus are expectedto be used in applications such as a medical soft member.

[0005] It is known that a composition and a structure of such a PHAproduced by a microorganism may considerably vary depending on the typeof a microorganism used for the production, a culture-medium compositionand culturing conditions. Investigations have been, therefore, mainlyfocused on controlling such a composition or structure for the purposeof improving physical properties of a PHA.

[0006] For example, Japanese Patent Application Laid-Open Nos. 6-15604,7-14352 and 8-19227 describe that Alcaligenes eutropus H16 (ATCC No.17699) and its variants may produce 3-hydroxybutyric acid (3HB) and itscopolymer with 3-hydroxyvaleric acid (3HV) with various compositionratios by changing a carbon source during culturing.

[0007] Japanese Patent Publication No. 2642937 discloses that PHA inwhich a monomer unit is 3-hydroxyalkanoate with 6 to 12 carbon atoms maybe produced by supplying a non-cyclic aliphatic hydrocarbon as a carbonsource to Pseudomonas oleovorans (ATCC No. 29347).

[0008] Japanese Patent Application Laid-Open No. 5-74492 disclosesmethods in which Methylobaterium sp., Paracoccus sp., Alcaligenes sp.,and Pseudomonas sp. are contacted with a primary alcohol with 3 to 7carbon atoms to produce a copolymer of 3HB and 3HV.

[0009] Japanese Patent Application Laid-Open Nos. 5-93049 and 7-265065disclose that Aeromonas caviae is cultured using oleic acid or olive oilas a carbon source to produce a two-component copolymer of 3HB and3-hydroxyhexanoic acid (3HHx).

[0010] Japanese Patent Application Laid-Open No. 9-191893 discloses thatComamonas acidovorans IF013852 is cultured using gluconic acid and1,4-butanediol as carbon sources to produce a polyester having 3HB and4-hydroxybutyric acid as monomer units.

[0011] Furthermore, it is reported that certain microorganisms producePHAs having a variety of substituents such as unsaturated hydrocarbon,ester, aryl (aromatic), and cyans groups, halogenated hydrocarbon andepoxide. Recently, there have been attempts for improving physicalproperties of a PHA produced by a microorganism using such a procedure.For example, Makromol. Chem., 191, 1957-1965 (1990); Macromolecules, 24,5256-5260 (1991); and Chirality, 3, 492-494 (1991) describe productionof a PHA comprising 3-hydroxy-5-phenylvaleric acid (3HPV) as a monomerunit by Pseudomonas oleovorans, where variations in polymer physicalproperties probably due to the presence of 3HPV were observed.

[0012] As described above, microorganism-produced PHAs with variouscombinations of composition and structure have been obtained by varyingfactors such as the type of a microorganism used, a culture mediumcomposition and culturing conditions. Each microorganism has anintrinsic PHA synthase with a substrate specificity which issignificantly different from others. Thus, it has been difficult toproduce PHAs comprising different monomer units suitable to a variety ofapplications using known microorganisms or PHA synthases in such knownmicroorganisms.

[0013] Meanwhile, as described above, a PHA having a variety ofsubstituents in its side chains may be expected to be a “functionalpolymer” having significantly useful functions and properties owing tothe properties of the introduced substituents. It is, therefore,extremely useful and important to search and develop a microorganismwhich can produce and store a very useful polymer having both suchfunctionality and biodegradability. Furthermore, identification of a PHAsynthase involved in production of the highly useful PHA and obtaining agene encoding the PHA synthase may allow us to produce a noveltransformed microorganism capable of producing a desired PHA. That is,constructing a recombinant vector comprising a gene encoding a PHAsynthase and providing a microorganism transformed by the recombinantvector may allow us to prepare a PHA using the transformed microorganismor to express a recombinant type of PHA synthase. As described above, itmay be important that a transformed microorganism is used to prepare adesired PHA for providing a highly useful tool for improving aproductivity for the PHA and for promoting utilization of the PHA.

SUMMARY OF THE INVENTION

[0014] Objects of this invention which can solve the above problems areto search a novel microorganism capable of producing and storing inmicroorganisms a PHA having a novel side-chain structure, to identify anenzyme protein related to the ability of producing the novel PHA, i.e.,a novel PHA synthase, and to determine a gene encoding its amino acidsequence. More specifically, an object of the present invention is toprovide a novel PHA synthase derived from a microorganism producing aPHA having a novel side chain structure and a DNA encoding its aminoacid sequence. Another object of this invention is to provide arecombinant vector to which a DNA encoding an available PHA synthase isintroduced and which is used for transformation of a microorganism and atransformed microorganism produced using the recombinant vector. Afurther object of this invention is to provide a process for expressingand producing a recombinant PHA synthase in the transformedmicroorganism and a process for preparing a desired PHA using thetransformed microorganism.

[0015] Still another object of this invention is to provide a modifiedPHA synthase in which its amino acid sequence is modified as long as anenzyme activity is not affected in expression of the recombinant PHAsynthase in the transformed microorganism as described above and a DNAencoding the modified amino acid sequence.

[0016] For developing a PHA having a novel side-chain structure usefulas, for example, a device material or a medical material aiming atsolving the above problems, the inventors have searched a novelmicroorganism capable of producing and storing the desired PHA therein.Additionally, the inventors have intensely investigated selected novelmicroorganisms producing a novel PHA for identifying a PHA synthaseinvolved in production of the novel PHA and for obtaining a geneencoding the PHA synthase. Furthermore, the inventors have conductedinvestigation for constructing a recombinant vector with a gene for theobtained PHA synthase, transforming a host microorganism using therecombinant vector, expressing a recombinant PHA synthase in thetransformed microorganism obtained and determining production of thedesired PHA.

[0017] In the course of the above investigation, the inventorssynthesized 5-(4-fluorophenyl) valeric acid (FPVA) represented byformula (II):

[0018] and separated from a soil a novel microorganism capable ofconverting the above compound (II) as a starting material (substrate)into corresponding 3-hydroxy-5-(4-fluorophenyl)valeric acid (3HFPV)represented by formula (III):

[0019] and producing and storing a novel PHA with a monomer unitrepresented by formula (I):

[0020] derived from 3HFPV. The novel microorganism separated isdesignated as P161 strain. The inventors have also found that inaddition to the above enzymatic activity for converting FPVA into 3HFPV,the P161 strain may also use 4-phenoxybutyric acid (PxBA) represented byformula (IV):

[0021] as a starting material (substrate) to convert it into3-hydroxy-4-phenoxybutyric acid (3HPxB) represented by formula (V):

[0022] and to produce and store a PHA with a monomer unit represented byformula (VI):

[0023] derived from 3HPxB. There have been no reports for microbialproduction of a PHA comprising 3HPxB as a monomer unit using PxBA as asubstrate or for microbial production of a PHA comprising 3HPxB as asole phenoxy-containing monomer unit.

[0024] An example of a microorganism capable of producing and storing aPHA with a monomer unit represented by formula (VI) derived from 3HPxBusing a substrate other than PxBA is Pseudomonas oleovorans using8-phenoxyoctanoic acid (PxOA) as a substrate described inMacromolecules, 29, 3432-3435, 1996. In Pseudomonas oleovorans,8-phenoxyoctanoic acid (PxOA) is used as a substrate, which is totallydifferent from the enzymatic reaction in P161 strain where PxBA is usedas a substrate to produce a PHA with a monomer unit represented byformula (VI) derived from a corresponding 3HPxB. In addition, for thecomposition of a PHA produced, the reported process using Pseudomonasoleovorans provides a copolymer consisting of three monomer units, i.e.,3-hydroxy-8-phenoxyoctanoic acid corresponding to PxOA as a substrate,3-hydroxy-6-phenoxyhexanoic acid as a byproduct derived from ametabolite of the substrate, and the desired 3HPxB. On the other hand, aprocess where P161 strain acts on the substrate PxBA provides a PHA with3HPxB derived from PxBA as a sole phenoxy-containing monomer unit.Taking the compositions of the PHAs also into consideration, it seemsthat there is fundamental difference in substrate specificity of a PHAsynthase between Pseudomonas oleovorans used in the above processreported and P161 strain. That is, a PHA synthase produced by P161strain is more preferable for production of a PHA with 3HPxB as amonomer unit.

[0025] Furthermore, the inventors have found that P161 strain can use6-phenylhexanoic acid (PHxA) represented by formula (VII):

[0026] as a starting material (substrate) to convert it intocorresponding 3-hydroxy-6-phenylhexanoic acid (3HPHx) represented byformula (VIII):

[0027] and to produce and store a novel PHA with a monomer unitrepresented by formula (IX):

[0028] derived from 3HPHx.

[0029] Microbiological properties of P161 strain are as follows.

[0030] <Microbiological Properties of P161 Strain>

[0031] Morphologic Properties

[0032] Cell shape and size: Sphere, φ0.6 μm

[0033] Bacilliform, 0.6 μm×1.5 to 2.0 μm

[0034] Cell polymorphism: Yes (elongation)

[0035] Motility: Yes

[0036] Sporulation: No

[0037] Gram stainability: Negative

[0038] Colonization: Circular, smooth in the overall periphery, lowconvex, smooth surface, pale yellow

[0039] Physiological Properties

[0040] Catalase: Positive

[0041] Oxidase: Positive

[0042] O/F test: oxidized form

[0043] Reduction of a nitrate: Positive

[0044] Indole formation: Negative

[0045] Acidification of dextrose: Negative

[0046] Arginine dihydrolase: Positive

[0047] Urease: Negative

[0048] Esculin hydrolysis: Negative

[0049] Gelatin hydrolysis: Negative

[0050] β-Galactosidase: Negative

[0051] Fluorochrome production on King's B agar: Positive

[0052] Substrate Assimilation Ability

[0053] Dextrose: Positive

[0054] L-Arabinose: Positive

[0055] D-Mannose: Positive

[0056] D-Mannitol: Positive

[0057] N-Acetyl-D-glucosamine: Positive

[0058] Maltose: Negative

[0059] Potassium gluconate: Positive

[0060] n-Capric acid: Positive

[0061] Adipic acid: Negative

[0062] dl-Malic acid: Positive

[0063] Sodium citrate: Positive

[0064] Phenyl acetate: Positive

[0065] From these microbiological properties, the inventors haveattempted to categorize P161 strain according to Bergey's Manual ofSystematic Bacteriology, Volume 1 (1984) and Bergey's Manual ofDeterminative Bacteriology 9th ed. (1994) to determine that the strainbelongs to Pseudomonas sp. Its taxonomic position could not beendetermined from these microbiological properties.

[0066] Thus, for categorizing P161 strain from its genetic properties,the inventors sequenced its 16S rRNA (SEQ ID NO. 5) and compared itshomology with the sequence of a 16S rRNA in a known Pseudomonas sp.microorganism. The results indicate quite higher homology in a 16S rRNAsequence between P161 strain and a known Pseudomonas jessenii.Furthermore, microbiological properties described for the knownPeudomonas jessenii in System. Appl. Microbiol., 20, 137-149 (1997) andSystem. Appl. Microbiol., 22, 45-58 (1999) was compared with those forP161 strain and observed considerable homology. From these results, itwas judged to be proper to categorize P161 strain in Pseudomonasjessenii, and thus it is designated as Pseudomonas jessenii P161. Therehave been no reports on a strain in Pseudomonas jessenii capable ofproducing a PHA as exhibited by P161 strain. The inventors have,therefore, determined that P161 strain is a novel microorganism. Theapplicant deposited Pseudomonas jessenii P161 to Patent MicroorganismDepository Center in the National Institute of Bioscience and HumanTechnology, Agency of Industrial Science and Technology, Ministry ofInternational Trade and Industry, under the deposition number of FERMP-17445. P161 strain has been internationally deposited on the basis ofthe Budapest Treaty, and its international accession number is “FERMBP-7376”.

[0067] The inventors achieved cloning a gene for a PHA synthase from thenovel microorganism P161 strain and sequenced the gene. The inventorsalso determined an amino acid sequence for the PHA synthase encoded bythe gene. Based on the above observation, the present invention wasachieved.

[0068] Specifically, a PHA synthase of the present invention is apolyhydroxyalkanoate synthase having an amino acid sequence of SEQ IDNO. 1 or 3. Furthermore, the PHA synthase of the present invention maybe a PHA synthase substantially retaining the amino acid sequence of SEQID NO. 1 and having a modified amino acid sequence where amino acids aredeleted, substituted or added as long as it does not deteriorate anactivity as the polyhydroxyalkanoate synthase, or a PHA synthasesubstantially retaining the amino acid sequence of SEQ ID NO. 3 andhaving a modified amino acid sequence where amino acids are deleted,substituted or added as long as it does not deteriorate activity as thepolyhydroxyalkanoate synthase.

[0069] A PHA synthase gene of the present invention is a gene for apolyhydroxyalkanoate synthase comprising a DNA encoding the amino acidsequence of SEQ ID NO. 1 or the sequence of its modified amino acid, ora gene for a polyhydroxyalkanoate synthase comprising a DNA encoding theamino acid sequence of SEQ ID NO. 3 or the sequence of its modifiedamino acid. Embodiments of a PHA synthase gene of the present inventionderived from a genome gene in P161 strain include a PHA synthase genecomprising a DNA sequence of SEQ ID NO. 2 as a DNA encoding the aminoacid sequence of SEQ ID NO. 1 and a PHA synthase gene comprising a DNAsequence of SEQ ID NO. 4 as a DNA encoding the amino acid sequence ofSEQ ID NO. 3.

[0070] This invention also provides a recombinant vector comprising agene DNA encoding the above amino acid sequence as apolyhydroxyalkanoate synthase gene. This invention also provides atransformed microorganism transformed by introducing a recombinantvector adapted to a host.

[0071] The present invention also provides a process for preparing apolyhydroxyalkanoate comprising the steps of culturing the transformedmicroorganism to which a recombinant vector has been introduced in aculture medium containing a substrate for a polyhydroxyalkanoatesynthase and collecting the polyhydroxyalkanoate from the culturepreparation. The present invention also provides a process for producinga polyhydroxyalkanoate comprising the steps of culturing the transformedmicroorganism to which a recombinant vector has been introduced andmaking the transformed microorganism produce the polyhydroxyalkanoate.

[0072] A preferable process for producing a polyhydroxyalkanoate mayutilize substrate specificity characteristic of a polyhydroxyalkanoatesynthase derived from P161 strain: for example, preparation of apolyhydroxyalkanoate comprising a monomer unit represented by formula(I) derived from 3HFPV utilizing the above transformed microorganism;preparation of a polyhydroxyalkanoate comprising a monomer unitrepresented by formula (VI) derived from 3HPxB, or preparation of apolyhydroxyalkanoate comprising a monomer unit represented by formula(IX) derived from 3HPHx.

[0073] A PHA synthase and a gene encoding the PHA synthase of thepresent invention are derived from a novel microorganism, Pseudomonasjessenii P161 strain and exhibits such substrate specificity that itselectively produces a PHA comprising a monomer unit having a novel sidechain structure. A recombinant vector comprising the PHA synthase geneand a microorganism transformed by the recombinant vector are capable ofproducing a PHA exhibiting substrate specificity similar to Pseudomonasjessenii P161. Thus, a PHA synthase gene of this invention encodes anenzyme which permits preparation of a PHA selectively comprising amonomer unit having a novel side-chain structure and allows us to createa transformed microorganism useful for preparing a PHA having varioususeful physical properties which may be expected to be applied to afunctional polymer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0074]FIG. 1 illustrates an amino acid sequence of the first PHAsynthase derived from Pseudomonas jessenii P161 (FERM BP-7376);

[0075]FIG. 2 illustrates an amino acid sequence of the first PHAsynthase derived from Pseudomonas jessenii P161 (FERM BP-7376);

[0076]FIG. 3 illustrates an amino acid sequence of the second PHAsynthase derived from Pseudomonas jessenii P161 (FERM BP-7376); and

[0077]FIG. 4 illustrates an amino acid sequence of the second PHAsynthase derived from Pseudomonas jessenii P161 (FERM BP-7376).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0078] A PHA synthase of this invention is an enzyme protein derivedfrom a novel microorganism isolated by the present inventors,Pseudomonas jessenii P161 (FERM BP-7376). Specifically, it can covert5-(4-fluorophenyl)valeric acid (FPVA) into corresponding3-hydroxy-5-(4-fluorophenyl)valeric acid (3HFPV), 4-phenoxybutyric acid(PxBA) into corresponding 3-hydroxy-4-phenoxybutyric acid (3HPxB) or6-phenylhexanoic acid (PHXA) into corresponding3-hydroxy-6-phenylhexanoic acid (3HPHx) and thus has enzymatic activityinvolved in production of a PHA comprising a corresponding monomer unit.

[0079] A PHA synthase and a gene encoding the enzyme of this inventionwill be more specifically described.

[0080] From P161 strain, the inventors have cloned a gene translatedinto a PHA synthase which exhibits the above substrate specificity, todetermine the presence of a PHA synthase comprising at least two aminoacid sequences. Specifically, a PHA synthase of this invention in achromogene in P161 strain comprises two enzymes, i.e., a PHA synthasecomprising the amino acid sequence of SEQ ID NO. 1 encoded by a DNAhaving the sequence of SEQ ID NO. 2 and a PHA synthase comprising theamino acid sequence of SEQ ID NO. 3 encoded by a DNA having the sequenceof SEQ ID NO. 4. Gene DNAs of the sequences of SEQ ID NOs. 2 and 4 maybe cloned by the following procedure.

[0081] Since a PHA synthase is an enzyme protein translated from achromogene, a chromosome DNA containing a desired PHA synthase is firstobtained. A chromosome DNA may be separated from P161 strain cells by aknown separation method. For example, P161 strain is cultured in a LBmedium or an M9 medium supplemented with an appropriate carbon source,disrupt and treated as described by, for example, Marmer et al. inJournal of Molecular Biology, Vol. 3, p. 208 (1961) to prepare achromosome DNA.

[0082] Then, a gene library is prepared from the chromosome DNA thusobtained. The chromosome DNA is degraded using an appropriaterestriction enzyme (e.g., Sau3AI) and a fragment with a proper length isligated with a ligatable vector truncated with a restriction enzyme(e.g., BamHI) to prepare a gene library.

[0083] Depending on a vector used in preparing a library, a properfragment length varies, e.g., about 4000 to 25000 bps for a usualplasmid vector and about 15000 to 30000 bps for a cosmid or phagevector. A proper length of DNA fragment may be collected by a knownmethod such as a method using a sucrose density gradient or using anagarose gel described in Molecular Cloning, Cold Spring HarborLaboratory (1982).

[0084] Since E. coli is used as a host microorganism in a gene library,a vector is a phage vector or plasmid vector which can autonomously growin the host microorganism (E. coli). Examples of phage or cosmic vectorsgenerally used include pWE15, M13, λEMBL3, λEMBL4, λFIXII, λDASHII,λZAPII, λgt10, λgt11, Charon4A and Charon21A. Examples of frequentlyused plasmid vectors include pBR, pUC, pBluescriptII, pGEM, pTZ and pETgroups. In addition to E. coli, various shuttle vectors may be used,e.g., vectors which may automonouosly grow in a plurality of hostmicroorganisms such as Pseudomonas sp. Again, these vectors may be,depending on a chromosome DNA to be ligated to them, truncated with aproper restriction enzyme to provide a desired fragment.

[0085] A chromosome DNA fragment may be ligated with a vector fragmentusing a DNA ligase. For example, a commercially available ligation kit(Takara Shuzo Co., Ltd., etc.) may be used. Thus, for example, variouschromosome DNA fragments may be ligated with a plasmid vector fragmentto prepare a mixture of recombinant plasmids comprising various DNAfragments (hereinafter, referred to as a “gene library”).

[0086] In addition to a method using a proper length of chromosome DNAfragment, a gene library may be prepared by a method that all mRNAs areextracted from P161 strain, purified and used for preparation of a cDNAfragment using a reverse transcriptase as described in MolecularCloning, Cold Spring Harbor Laboratory, 1982. Alternatively, a preparedvector is used in a gene library to transform or transduce to E. coli,and then the host E. coli is cultured to amplify the gene library to alarge amount as described in Molecular Cloning, Cold Spring HarborLaboratory, 1982.

[0087] A recombinant vector comprising a gene DNA fragment may beintroduced into a host microorganism by a known method. For example,when using E. coli as a host microorganism, a recombinant plasmid vectormay be introduced using a calcium chloride method (Journal of MolecularBiology, Vol. 53, p. 159 (1970)), a rubidium chloride method (Methods inEnzymology, Vol. 68, p. 253 (1979)), electroporation (Current Protocolsin Molecular Biology, Vol. 1, p. 1.8.4 (1994)). When using a cosmidvector or phage vector, transduction in a host E. coli may be conductedusing in vitro packaging (Current Protocols in Molecular Biology, Vol.1, p. 5.7.1 (1994)). Alternatively, conjugational transfer with a strainretaining a recombinant vector may be utilized to prepare a strainretaining a vector.

[0088] Then, from the gene library, a probe is prepared for obtaining aDNA fragment comprising a PHA synthase gene of P161 strain.

[0089] Some base sequences have been reported for PHA synthase genes inknown microorganisms; for example, Peoples, O. P. and Sinskey, A. J., J.Biol. Chem., 264, 15293 (1989); Huisman, G. W. et al., J. Biol. Chem.,266, 2191 (1991); Pieper, U. et al., FEMS Microbiol. Lett., 96, 73(1992); Timm, A. and Steinbuchel, A., Eur. J. Biochem., 209, 15(1992);Matsusaki, H. et al., J. Bacteriol., 180, 6459 (1998). These reportedsequences are compared to select a region where a sequence is preservedto a higher degree and thus to design an oligonucleotide for a primerused in polymerase chain reaction (hereinafter, referred to as “PCR”).Such oligonucleotides for a primer utilizing a common feature of PHAsynthase genes include, but not limited to, a sequence described inTimm, A. and Steinbuchel, A., Eur. J. Biochem., 209, 15 (1992). Anoligonucleotide may be synthesized using, for example, a commerciallyavailable DNA synthesizer such as Custom Synthesis Service,Amersham-Pharmacia Biotech, depending on a designed sequence.

[0090] For a PHA synthase gene derived from P161 of this invention,synthetic DNAs having the sequences of SEQ ID NOs. 6 and 7 weredesigned.

[0091] Then, the designed oligonucleotide as a primer is subject topolymerase chain reaction (PCR) using a chromosome DNA in P161 strain asa template to obtain a PCR amplified fragment. The PCR amplifiedfragment, which is derived from the primer, comprises a sequence commonin PHA synthase genes at both ends. A partial sequence derived from thePHA synthase gene itself in P161 strain as a template is containedbetween sequences complementary to the primer at both ends.

[0092] The PCR amplified fragment obtained is, therefore, almost 100%homologous to the PHA synthase gene in P161 strain and is expected toexhibit a higher S/N ratio as a probe in colony hybridization. Inaddition, it may facilitate stringency control of hybridization.

[0093] The above PCR amplified fragment is labeled with an appropriatereagent and used as a probe to colony-hybridize the above chromosome DNAlibrary for selecting a recombinant E. coli strain retaining the PHAsynthase gene (Current Protocols in Molecular Biology, Vol. 1, p. 6.0.3(1994)). For example, the PCR amplified fragment may be labeled using acommon detection system using a labeled enzyme or a commerciallyavailable kit such as AlkPhosDirect (Amersham-Pharmacia Biotech).

[0094] A recombinant E. coli strain retaining a gene fragment comprisinga PHA synthase gene may be selected by, in addition to the above methodusing a gene type, a method using a phenotype where PHA synthesis isdirectly evaluated. Specifically, in expression of a PHA synthase from aretained PHA synthase gene in a recombinant E. coli strain, PHA isproduced by the PHA synthase. PHA synthesis may be detected to select arecombinant E. coli strain in which the PHA synthase is expressed. PHAsynthesis may be detected by, for example, staining with Sudan Black B(Archives of Biotechnology, Vol. 71, p. 283 (1970)) or determination ofPHA accumulation by phase contrast microscopy.

[0095] A plasmid is collected from a recombinant E. coli selected by anyof the above methods using an alkali method (Current Protocols inMolecular Biology, Vol. 1, p. 1.6.1 (1994)). The collected plasmid maybe used to provide a DNA fragment comprising a PHA synthase gene ormultiple DNA fragments partially containing a PHA synthase gene. The DNAfragment obtained may be sequenced by, for example, the Sanger'ssequencing method (Molecular Cloning, Vol. 2, p. 13.3 (1989).Specifically, it may be conducted by a dye-primer method or adye-terminator method using an automatic sequencer such as DNA Sequencer377A (Parkin Elmer). Since the sequence of the vector itself in whichthe DNA fragment has been incorporated is known, the sequence of the DNAfragment cloned therein may be unequivocally analyzed.

[0096] After sequencing all the obtained DNA fragments comprising a PHAsynthase gene, hybridization may be conducted using a DNA fragmentprepared by an appropriate method such as chemical synthesis, PCR usinga chromosome DNA as a template or degradation of a DNA fragmentcomprising the sequence with a restriction enzyme as a probe to providea PHA synthase gene DNA of this invention.

[0097] The inventors have selected a gene translated into a PHA synthaseexhibiting the above substrate specificity from P161 strain according tothe above procedure to find a PHA synthase comprising at least two aminoacid sequences. Specifically, the inventors have found a PHA synthasegene collected from the chromosome DNA of P161 strain and comprising thesequence of SEQ ID NO. 2 and a PHA synthase encoded by the gene andcomprising the amino acid of SEQ ID NO. 1 as well as a PHA synthase genecomprising the sequence of SEQ ID NO. 4 and a PHA synthase encoded bythe gene and comprising the amino acid of SEQ ID NO. 3.

[0098] A PHA synthase gene of the present invention may include adegenerated isomer encoding the same polypeptide which has the sameamino acid sequence and is different in a degeneration codon. Morespecifically, it also includes a degenerated isomer by selection andconversion of a more frequently used degenerated codon encoding the sameamino acid depending on a host. Besides the PHA synthase comprising theamino acid sequence of SEQ ID NO. 1 inherent in P161 strain and the PHAsynthase comprising the amino acid sequence of SEQ ID NO. 3, a PHAsynthase of this invention may have mutation such as deletion,substitution and addition for several amino acids as long as its PHAproducing activity and substrate specificity may not be deteriorated orthe amino acid sequence may be maintained. Mutation such as deletion,substitution and addition may be introduced by a site mutationintroduction technique based on a PHA synthase gene inherent in P161strain having the sequence of SEQ ID NO. 2 or 4 (Current Protocols inMolecular Biology Vol. 1, p. 8.1.1 (1994)).

[0099] A recombinant vector of the present invention is used in anapplication where a recombinant PHA synthase of this invention isexpressed using Pseudomonas sp. or a microorganism such as E. coli as ahost. It is, therefore, preferable that the recombinant vector of thisinvention itself can autonomously replicate in a host used whilecomprising a promoter for expression, a PHA synthase gene DNA of thisinvention and a transcription termination sequence suitable to the host.In addition, it is preferable that after introducing the recombinantvector, a vector comprising various marker genes used for its selectionis used.

[0100] Expression vectors suitable to various types of bacterial hostssuch as Pseudomonas sp. and E. coli include pLA2917 (ATCC 37355) havinga RK2 replication origin which may be replicated and retained by a rangeof hosts or pJRD215 (ATCC 37533) having a RSF1010 replication origin.Without being limited to these, any vector having a replication originwhich may be replicated and retained by a range of hosts may be used.Any promoter which may be expressed in a bacterium as a host may beused; for example, promoters derived from E. coli, a phage, etc. such astrp, trc, tac, lac, PL, PR, T7 and T3 promoters.

[0101] When using a yeast as a host, an expression vector may be YEp13,YCp50, pRS or pYEX vector. A promoter may be, for example, GAL or AODpromoter.

[0102] A transformed microorganism of this invention may be produced byintroducing a recombinant vector of this invention into a host suitableto an expression vector used during preparing the recombinant vector.Examples of bacteria which may be used as a host include Esherichia sp.,Pseudomonas sp., Ralstonia sp., Alcaligenes sp., Comamonas sp.,Burkholderia sp., Agrobacterium sp., Flabobacterium sp., Vibrio sp.,Enterobacter sp., Rhizobium sp., Gluconobacter sp., Acinetobacter sp.,Moraxella sp., Nitrosomonas sp., Aeromonas sp., Paracoccus sp., Bacillussp., Clostridium sp., Lactobacillus sp., Corynebacterium sp.,Arthrobacter sp., Achromobacter sp., Micrococcus sp., Mycobacterium sp.,Streptococcus sp., Streptomyces sp., Actinomyces sp., Norcadia sp. andMethylobacterium sp. A recombinant DNA may be introduced into abacterium by an appropriate technique such as the above calcium chloridemethod and electroporation.

[0103] Besides the above bacteria, yeasts and molds such asSaccharomyces sp. and Candida sp. may be used as a host. A recombinantDNA may be introduced into an yeast by, for example, electroporation(Methods Enzymol., 194, 182-187 (1990)), a spheroplast method (Proc.Natl. Acad. Sci. USA, 84, 1929-1933 (1978)) and a lithium acetate method(J. Bacteriol., 153, 163-168 (1983)).

[0104] A PHA synthase of this invention may be prepared by culturing atransformant of this invention prepared by the above procedure andmaking a corresponding PHA synthase gene in an introduced expressionvector producing the synthase as a recombinant protein. The PHA synthaseof this invention is produced and accumulated in the culture (culturedbacterium or culture supernatant) and separated from the culture to beused for production of a recombinant enzyme protein. For this purpose, atransformant of this invention may be cultured by a usual procedure usedfor culturing a host. Culturing may be conducted by any of commonmethods used for culturing a microorganism such as batch, flow batch,continuous culturing and reactor styles. This culturing may be conductedby using, for example, a medium containing an inducer for expressing theabove polyhydroxyalkanoate synthase gene.

[0105] For a transformant obtained using a bacterium such as E. coli asa host, a medium used for culturing may be a complete medium orsynthetic medium such as LB medium and M9 medium. A microorganism may begrown by aerobically culturing at a culturing temperature of 25 to 37°C. for 8 to 72 hours. Then, the bacteria are collected for obtaining aPHA synthase accumulated in them. Examples of a carbon source for themicroorganism include sugars such as glucose, fructose, sucrose,maltose, galactose and starches; lower alcohols such as ethanol,propanol and butanol; polyalcohols such as glycerol; organic acids suchas acetic acid, citric acid, succinic acid, tartaric acid, lactic acidand gluconic acid; and aliphatic acids such as propionic acid, butanoicacid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid,nonanoic acid, decanoic acid, undecanoic acid and dodecanoic acid.

[0106] Examples of a nitrogen source include ammonia; ammonium saltssuch as ammonium chloride, ammonium sulfate and ammonium phosphate; andnatural product derivatives such as peptone, meat extract, yeastextract, malt extract, casein decomposition products and corn steepliquor. Examples of an inorganic material include potassium dihydrogenphosphate, potassium monohydrogen phosphate, magnesium phosphate,magnesium sulfate and sodium chloride. The culture medium may contain anantibiotic such as kanamycin, ampicillin, tetracyclin, chloramphenicoland streptomycin, depending on, for example, the type of a drugresistance gene used as a marker gene.

[0107] When using an inducible promoter in an expression vector,expression may be enhanced by adding a proper inducer depending on thetype of the promoter during culturing a transformed microorganism. Forexample, the inducer may be isopropyl-β-D-thiogalactopyranoside (IPTG),tetracyclin or indoleacrylic acid (IAA).

[0108] A PHA synthase may be separated and purified by centrifuging andcollecting a culture obtained and processing it by a technique such asaffinity chromatography, cation or anion exchange chromatography and gelfiltration alone or in combination as appropriate. Whether a purifiedmaterial is a desired enzyme is determined by a usual method such as SDSpolyacrylamide gel electrophoresis and Western blotting.

[0109] This invention is not limited to the procedures as describedabove for culturing of a transformed microorganism of this invention,production of a PHA synthase by the transformed microorganism of thisinvention and accumulating it in bacterial cells, and collection andpurification of the PHA synthase from the cells.

[0110] A transformed microorganism of this invention may be used forexpressing a recombinant PHA synthase to produce a desired PHA. Forexample, the microorganism may be cultured under the above culturingconditions to produce a recombinant PHA synthase while a substratecorresponding to the desired PHA on which the PHA synthase acts is addedto a medium. Most conveniently, the PHA may be collected from theculture and the producing bacteria by extraction with an organic solventcommonly used such as chloroform. In an environment where using anorganic solvent such as chloroform is undesirable, the culture may betreated a surfactant such as SDS, an enzyme such as lysozyme, or anagent such as EDTA, sodium hypochlorite and ammonia to remove bacteriumcomponents other than the PHA for collecting the PHA. This invention isnot limited to the above procedures for culturing of a transformedmicroorganism of this invention for production of a PHA, production of aPHA by and accumulation thereof in a cultured microorganism, andcollection of the PHA from a recombinant microorganism.

EXAMPLES

[0111] This invention will be more specifically described with referenceto Examples, although these Examples are illustrated as the bestembodiments of this invention and do not limit the technical range ofthis invention.

Example 1

[0112] Cloning of a PHA Synthase Gene of P161 Strain

[0113] P161 strain was cultured in 100 mL of LB medium (1% polypeptone,0.5% yeast extract, 0.5% sodium chloride, pH 7.4) at 30° C. overnightand then a chromosome DNA was separated and collected as described byMarmer. The obtained chromosome DNA was completely digested using arestriction enzyme BglII. A vector pUC18 was cleaved with a restrictionenzyme BamHI. After dephosphorylation of the terminals (MolecularCloning, Vol. 1, p. 5.7.2 (1989), Cold Spring Harbor Laboratory), thecleaved site of the vector (cloning site) and the chromosome DNAfragment after BglII complete digestion were ligated using a DNAligation kit Ver. II (Takara Shuzo Co., Ltd.). The plasmid vector inwhich the chromosome DNA fragment was integrated was used to transformEscheichia coli HB101 for preparing a chromosome DNA library for P161strain.

[0114] Then, in order to select a DNA fragment comprising a PHA synthasegene of P161 strain, a probe for colony hybridization was prepared. Anoligonucleotide consisting of the sequences of SEQ ID NOs. 6 and 7(Amersham-Pharmacia Biotech) was prepared and used as a primer for PCRusing the chromosome DNA as a template. A PCR-amplified DNA fragment wasused as a probe. Labeling of the probe was conducted using acommercially available labeling enzyme system AlkPhosDirect(Amersham-Pharmacia Biotech). The labeled probe thus obtained was usedto select an E. coli strain containing a recombinant plasmid comprisingthe PHA synthase gene from the chromosome DNA library of P161 strain bycolony hybridization. From the selected strain, the plasmid wascollected by an alkali method to prepare a DNA fragment comprising a PHAsynthase gene.

[0115] The gene DNA fragment thus obtained was recombined in a vectorpBBR122 (Mo BiTec) comprising a wide host range of replication regionwhich did not belong to IncP, IncQ or IncW in an incompatible group. Therecombinant plasmid was transformed in Pseudomonas jessenii P161m1strain (a strain depleted of PHA synthesizing ability) byelectroporation, and then the P161m1 strain regained PHA synthesizingability and exhibited complementarity. It demonstrates that the selectedgene DNA fragment comprises a region of a PHA synthase gene translatableinto a PHA synthase in Pseudomonas jessenii P161m1.

[0116] The DNA fragment comprising a PHA synthase gene was sequenced bythe Sanger's sequencing method. It was thus found that the determinedsequence comprised the sequences of SEQ ID NOs. 2 and 4 each of whichencoded a peptide chain. As described below, it was determined that bothproteins consisting of a peptide chain had enzyme activity and that thesequences of SEQ ID NOs. 2 and 4 were therefore PHA synthase genes.Specifically, it was found that the sequences of SEQ ID NOs. 2 and 4encoded the amino acid sequences of SEQ ID NOs. 1 and 3, respectively,and that a protein comprising one of these amino acid sequences alonecould produce a PHA.

Example 2

[0117] Recombination of a PHA Synthase Gene of P161 Strain to anExpression Vector

[0118] A PHA synthase gene having the sequence of SEQ ID NO. 2 was PCRedusing a chromosome DNA as a template to reproduce the whole length of aPHA synthase gene. An oligonucleotide having a sequence which was anupstream primer to the sequence of SEQ ID NO. 2 and had a sequenceupstream of its initiation codon (SEQ ID NO. 8) and an oligonucleotidehaving a sequence which was a downstream primer to the sequence of SEQID NO. 2 and had a sequence downstream of its termination codon (SEQ IDNO. 9) were designed and prepared (Amersham-Pharmacia Biotech). Usingthese oligonucleotides as a primer, PCR was conducted to amplify thewhole length of the PHA synthase gene (LA-PCR kit; Takara Shuzo Co.,Ltd.).

[0119] Likewise, a PHA synthase gene having the sequence of SEQ ID NO. 4was PCRed using a chromosome DNA as a template to reproduce the wholelength of a PHA synthase gene. An oligonucleotide having a sequencewhich was an upstream primer to the sequence of SEQ ID NO. 4 and had asequence upstream of its initiation codon (SEQ ID NO. 10) and anoligonucleotide having a sequence which was a downstream primer to thesequence of SEQ ID NO. 4 and had a sequence downstream of itstermination codon (SEQ ID NO. 11) were designed and prepared(Amersham-Pharmacia Biotech). Using these oligonucleotides as a primer,PCR was conducted to amplify the whole length of the PHA synthase gene(LA-PCR kit; Takara Shuzo Co., Ltd.).

[0120] Each of the obtained PCR amplified fragment containing the wholelength of the PHA synthase gene was completely digested using arestriction enzyme HindIII. Separately, an expression vector pTrc99A wasalso truncated with a restriction enzyme HindIII and dephosphorylated(Molecular Cloning, Vol. 1, p. 5.7.2 (1989), Cold Spring HarborLaboratory). To the truncated site of the expression vector pTrc99A wasligated the DNA fragment comprising the whole length of the PHA synthasegene from which unnecessary sequences had been removed at both ends,using a DNA ligation kit Ver. II (Takara Shuzo Co., Ltd.).

[0121] Using the recombinant plasmids obtained, Escherichia coli HB101(Takara Shuzo Co., Ltd.) was transformed by a calcium chloride method.The recombinants were cultured, and the recombinant plasmids wereamplified and collected individually. The recombinant plasmid retainingthe gene DNA of SEQ ID NO. 2 was designated pP161-C1 (derived from SEQID NO. 2) while the recombinant plasmid retaining the gene DNA of SEQ IDNO. 4 was designated pP161-C2 (derived from SEQ ID NO. 4).

Example 3

[0122] PHA Production (1) Using a PHA Synthase Gene Recombinant E. coli

[0123] Using the recombinant plasmids obtained in Example 2, pP161-C1(derived from SEQ ID NO. 2) and pP161-C2 (derived from SEQ ID NO. 4), anEscherichia coli HB101fB (fadB deficient strain) was transformed by acalcium chloride method to prepare recombinant E. coli strains retainingthe recombinant plasmid, pP161-C1 and pP161-C2 recombinant strains,respectively.

[0124] Each of the pP161-C1 and pP161-C2 recombinant strains wasinoculated to 200 mL of M9 medium containing 0.5% yeast extract and 0.1%FPVA, and the medium was shaken at 37° C. with a rate of 125strokes/min. After 24 hours, the cells were collected by centrifugation,washed once with cold methanol and lyophilized.

[0125] The lyophilized pellet was suspended in 100 mL of chloroform andthe suspension was stirred at 60° C. for 20 hours to extract a PHA.After filtering the extract through a membrane filter with a pore sizeof 0.45 μm, the filtrate was concentrated by rotary evaporation. Then,the concentrate was re-suspended in cold methanol and the precipitantwas collected and dried in vacuo to provide a PHA. The PHA thus obtainedwas subject to methanolysis as usual and analyzed using a gaschromatography-mass spectrometry apparatus (GC-MS, Shimadzu QP-5050, EItechnique) to identify methyl-esterified PHA monomer units. Table 1shows together a cell dry weight, a polymer dry weight for a collectedPHA, a polymer yield per a cell (polymer dry weight/cell dry weight) andidentities of monomer units for each strain. TABLE 1 pP161-C1recombinant pP161-C2 recombinant strain strain Cell dry weight 900 mg/L940 mg/L Polymer dry weight  35 mg/L  37 mg/L Polymer dry weight/Celldry weight 4% 4% Monomer unit composition (area ratio) 3-Hydroxybutyricacid 0% 0% 3-Hydroxyvaleric acid 0% 0% 3-Hydroxyhexanoic acid 0% 0%3-Hydroxyheptanoic acid 7% 5% 3-Hydroxyoctanoic acid 6% 5%3-Hydroxynonanoic acid 9% 12%  3-Hydroxydecanoic acid 12%  12% 3-Hydroxy-5-(4-fluorophenyl) 66%  66%  valeric acid

[0126] These results show that both pP161-C1 and pP161-C2 recombinantstrains produce, from the substrate 5-(4-fluorophenyl) valeric acid,PHAs comprising a monomer unit represented by formula (I) derived fromcorresponding 3-hydroxy-5-(4-fluorophenyl) valeric acid as a maincomponent. It is, therefore, demonstrated that although the pP161-C1 andpP161-C2 recombinant strains exclusively produce PHA synthases havingthe amino acid sequences of SEQ ID NOs. 1 and 3 translated from the PHAsynthase genes comprising the sequences of SEQ ID NOs. 2 and 4,respectively, both strains similarly convert the substrate5-(4-fluorophenyl) valeric acid into the monomer unit represented byformula (I) derived from corresponding 3-hydroxy-5-(4-fluorophenyl)valeric acid and produce a PHA containing the monomer unit.

Example 4

[0127] PHA Production (2) Using a PHA Synthase Gene Recombinant E. coli

[0128] Each of the pP161-C1 and pP161-C2 recombinant strains wasinoculated to 200 mL of M9 medium containing 0.5% yeast extract and 0.2%4-phenoxybutyric acid(PxBA), and the medium was shaken at 37° C. with arate of 125 strokes/min. After 24 hours, the cells were collected bycentrifugation, washed once with cold methanol and lyophilized.

[0129] The lyophilized pellet was suspended in 100 mL of chloroform andthe suspension was stirred at 60° C. for 20 hours to extract a PHA.After filtering the extract through a membrane filter with a pore sizeof 0.45 μm, the filtrate was concentrated by rotary evaporation. Then,the concentrate was re-suspended in cold methanol and the precipitantwas collected and dried in vacuo to provide a PHA. The PHA thus obtainedwas subject to methanolysis as usual and analyzed using a gaschromatography-mass spectrometry apparatus (GC-MS, Shimadzu QP-5050, EItechnique) to identify methyl-esterified PHA monomer units. Table 2shows together a cell dry weight, a polymer dry weight for a collectedPHA, a polymer yield per a cell (polymer dry weight/cell dry weight) andidentities of monomer units for each strain. TABLE 2 pP161-C1recombinant pP161-C2 recombinant strain strain Cell dry weight 750 mg/L720 mg/L Polymer dry weight  4 mg/L  4 mg/L Polymer dry weight/Cell dryweight 0.5% 0.5% Monomer unit composition (area ratio) 3-Hydroxybutyricacid 0% 0% 3-Hydroxyvaleric acid 0% 0% 3-Hydroxyhexanoic acid 0% 0%3-Hydroxyheptanoic acid 2% 2% 3-Hydroxyoctanoic acid 3% 3%3-Hydroxynonanoic acid 5% 7% 3-Hydroxydecanoic acid 5% 6%3-Hydroxy-4-phenoxybutyric acid 85%  82% 

[0130] These results show that both pP161-C1 and pP161-C2 recombinantstrains produce, from the substrate 4-phenoxybutyric acid, PHAscomprising a monomer unit represented by formula (VI) derived fromcorresponding 3-hydroxy-4-phenoxybutyric acid as a main component. Itis, therefore, demonstrated that although the pP161-C1 and pP161-C2recombinant strains exclusively produce PHA synthases having the aminoacid sequences of SEQ ID NOs. 1 and 3 translated from the PHA synthasegenes comprising the sequences of SEQ ID NOs. 2 and 4, respectively,both strains similarly convert the substrate 4-phenoxybutyric acid intothe monomer unit represented by formula (VI) derived from corresponding3-hydroxy-4-phenoxybutyric acid and produce a PHA containing the monomerunit.

Example 5

[0131] PHA Production (3) Using a PHA Synthase Gene Recombinant E. coli

[0132] Each of the pP161-C1 and pP161-C2 recombinant strains wasinoculated to 200 mL of M9 medium containing 0.5% yeast extract and 0.1%6-phenylhexanoic acid (PHxA), and the medium was shaken at 37° C. with arate of 125 strokes/min. After 24 hours, the cells were collected bycentrifugation, washed once with cold methanol and lyophilized.

[0133] The lyophilized pellet was suspended in 100 mL of chloroform andthe suspension was stirred at 60° C. for 20 hours to extract a PHA.After filtering the extract through a membrane filter with a pore sizeof 0.45 μm, the filtrate was concentrated by rotary evaporation. Then,the concentrate was re-suspended in cold methanol and the precipitantwas collected and dried in vacuo to provide a PHA. The PHA thus obtainedwas subject to methanolysis as usual and analyzed using a gaschromatography-mass spectrometry apparatus (GC-MS, Shimadzu QP-5050, EItechnique) to identify methyl-esterified PHA monomer units. Table 3shows together a cell dry weight, a polymer dry weight for a collectedPHA, a polymer yield per a cell (polymer dry weight/cell dry weight) andidentities of monomer units for each strain. TABLE 3 pP161-C1recombinant pP161-C2 recombinant strain strain Cell dry weight 1050 mg/L980 mg/L Polymer dry weight  73 mg/L  70 mg/L Polymer dry weight/Celldry weight 7% 7% Monomer unit composition (area ratio) 3-Hydroxybutyricacid 0% 0% 3-Hydroxyvaleric acid 0% 0% 3-Hydroxyhexanoic acid 0% 0%3-Hydroxyheptanoic acid 3% 3% 3-Hydroxyoctanoic acid 3% 4%3-Hydroxynonanoic acid 5% 2% 3-Hydroxydecanoic acid 5% 4%3-Hydroxy-6-phenylhexanoic 84%  87%  acid

[0134] These results show that both pP161-C1 and pP161-C2 recombinantstrains produce, from the substrate 6-phenylhexanoic acid, PHAscomprising a monomer unit represented by formula (IX) derived fromcorresponding 3-hydroxy-6-phenylhexanoic acid as a main component. Itis, therefore, demonstrated that although the pP161-C1 and pP161-C2recombinant strains exclusively produce PHA synthases having the aminoacid sequences of SEQ ID NOs. 1 and 3 translated from the PHA synthasegenes comprising the sequences of SEQ ID NOs. 2 and 4, respectively,both strains similarly convert the substrate 6-phenylhexanoic acid intothe monomer unit represented by formula (IX) derived from corresponding3-hydroxy-6-phenylhexanoic acid and produce a PHA containing the monomerunit.

[0135] The results together with those in Examples 3 and 4 demonstratethat the PHA synthases having the amino acid sequences of SEQ ID NOs. 1and 3 have enzyme activity mutually similar in substrate specificity.

Example 6

[0136] Homology of a PHAsynthase Gene of P161 Strain

[0137] In the same manner as in Example 1, the chromosome DNA of P161strain was separated and collected. Further, Pseudomonas olevoransATCC29347, Pseudomonas putida Tk2440 and Pseudomonas aeruginose PA01were cultured and the chromosome DNAs thereof were separated andcollected in the same as in Example 1.

[0138] Next, a probe for hybridization was prepared for confirminghomology of a PHAsynthase gene of P161 strain. In the same manner as inExample 2, an oligonucleotide having the sequences of SEQ ID NOs 7 and 8were synthesized. (Amersham-Pharmacia Biotech) PCR was conducted usingthe thus synthesized oligonucleotide as a primer and the chromosome DNAas a template. The obtained PCR amplified DNA fragments were used as aprobe. Labeling of the probe using AlkphosDirect (Amersham-PharmaciaBiotech) was conducted to obtain the labeled probe referred to as“phaC1”. In the same manner as above, an oligonucleotide having thesequences of SEQ ID NOs 9 and 10 were synthesized. (Amersham-PharmaciaBiotech) PCR was conducted using the thus synthesized oligonucleotide asa primer and the chromosome DNA as a template. Labeling of the obtainedPCR amplified DNA fragments was conducted in the same manner as above toobtain the labeled probe referred to as “phaC2”.

[0139] Using the thus obtained probes, homology of the PHA synthase genewas confirmed by a dot-blot method. The thus prepared chlomosome DNA wasalkalized and then blotted on a nylon film (Tropilon-45, produced byTropix Co.) by lpg at respective points using a dot-blot apparatus(BRL).

[0140] The film was dried at 80° C. for two hours, then put in a vinylbag. 3 ml of the solution for hybridization prepared according to therecipe of AlkPhosDirect was added thereto, and hybridization wasconducted at 55° C. for one hour. 15 ng of the labeled probe phaC1 orpheC2 per 3 ml of the above hybridization solution (5 ng/ml) was addedto the nylon film, and hybridization was conducted at 55° C. for 12hours. And then the nylon film was put out from the vinyl bag, andwashed two times for 10 minutes at 55° C. with a first washing bufferaccording the recipe of AlkPhosDirect. And then after it was washed twotimes for 5 minutes at room temperature with a second washing bufferaccording to the recipe of AlkPhosDirect, a detecting step was carriedout using CDP-Star attached to AlkPhosDirect according to the recipe.

[0141] Further, a detecting step was carried out under the sameconditions as above except that hybridization and first washing wereconducted at 60° C. or 65° C. The results were shown in Table 4. TABLE 4Temperature (° C.) 55 60 65 Probe phaC1 Target DNA P161 Strain A A BATCC29347 Strain C D D KT2440 Strain C D D PAO1 Strain D D D Probe phaC2Target DNA P91 Strain A A B ATCC29347 Strain C D D KT2440 Strain C D DPAO1 Strain D D D

[0142] A: Strong Signal, B: Signal, C: Slight Signal,

[0143] D: No Signal

1 11 1 559 PRT Pseudomonas jessenii P161 ; FERM BP-7376 1 Met Ser AsnLys Asn Asn Asp Asp Leu Lys Ser Gln Ala Ser Glu 1 5 10 15 Asn Thr LeuGly Leu Asn Pro Val Val Gly Leu Arg Gly Lys Asp 20 25 30 Leu Leu Ala SerAla Arg Met Val Leu Arg Gln Ala Ile Lys Gln 35 40 45 Pro Ile His Ser AlaArg His Val Ala His Phe Gly Leu Glu Leu 50 55 60 Lys Asn Val Leu Leu GlyLys Ser Glu Leu Leu Pro Thr Ser Asp 65 70 75 Asp Arg Arg Phe Ala Asp ProAla Trp Ser Gln Asn Pro Leu Tyr 80 85 90 Lys Arg Tyr Leu Gln Thr Tyr LeuAla Trp Arg Lys Glu Leu His 95 100 105 Asp Trp Ile Asp Asp Ser Asn LeuPro Ala Lys Asp Val Ser Arg 110 115 120 Gly His Phe Val Ile Asn Leu MetThr Glu Ala Phe Ala Pro Thr 125 130 135 Asn Thr Ala Ala Asn Pro Ala AlaVal Lys Arg Phe Phe Glu Thr 140 145 150 Gly Gly Lys Ser Leu Leu Asp GlyLeu Ser His Leu Ala Lys Asp 155 160 165 Leu Val His Asn Gly Gly Met ProSer Gln Val Asn Met Gly Ala 170 175 180 Phe Glu Val Gly Lys Thr Leu GlyVal Thr Glu Gly Ala Val Val 185 190 195 Phe Arg Asn Asp Val Leu Glu LeuIle Gln Tyr Lys Pro Ile Thr 200 205 210 Glu Gln Val His Glu Arg Pro LeuLeu Val Val Pro Pro Gln Ile 215 220 225 Asn Lys Phe Tyr Val Phe Asp LeuSer Pro Glu Lys Ser Leu Ala 230 235 240 Arg Phe Cys Leu Arg Asn Asn ValGln Thr Phe Ile Val Ser Trp 245 250 255 Arg Asn Pro Thr Lys Glu Gln ArgGlu Trp Gly Leu Ser Thr Tyr 260 265 270 Ile Glu Ala Leu Lys Glu Ala ValAsp Val Val Thr Ala Ile Thr 275 280 285 Gly Ser Lys Asp Val Asn Met LeuGly Ala Cys Ser Gly Gly Ile 290 295 300 Thr Cys Thr Ala Leu Leu Gly HisTyr Ala Ala Ile Gly Glu Asn 305 310 315 Lys Val Asn Ala Leu Thr Leu LeuVal Ser Val Leu Asp Thr Thr 320 325 330 Leu Asp Ser Asp Val Ala Leu PheVal Asp Glu Gln Thr Leu Glu 335 340 345 Ala Ala Lys Arg Gln Ser Tyr GlnAla Gly Val Leu Glu Gly Arg 350 355 360 Asp Met Ala Lys Val Phe Ala TrpMet Arg Pro Asn Asp Leu Ile 365 370 375 Trp Asn Tyr Trp Val Asn Asn TyrLeu Leu Gly Asn Glu Pro Pro 380 385 390 Val Phe Asp Ile Leu Phe Trp AsnAsn Asp Thr Thr Arg Leu Pro 395 400 405 Ala Ala Phe His Gly Asp Leu IleGlu Met Phe Lys Ser Asn Pro 410 415 420 Leu Thr Arg Ala Asp Ala Leu GluVal Cys Gly Thr Pro Ile Asp 425 430 435 Leu Lys Lys Val Thr Ala Asp IlePhe Ser Leu Ala Gly Thr Ser 440 445 450 Asp His Ile Thr Pro Trp Arg SerCys Tyr Lys Ser Ala Gln Leu 455 460 465 Phe Gly Gly Asn Val Glu Phe ValLeu Ser Ser Ser Gly His Ile 470 475 480 Gln Ser Ile Leu Asn Pro Pro GlyAsn Pro Lys Ser Arg Tyr Met 485 490 495 Thr Ser Thr Glu Met Pro Ala AsnAla Asp Asp Trp Gln Glu Glu 500 505 510 Ser Thr Lys His Ala Asp Ser TrpTrp Leu His Trp Gln Ala Trp 515 520 525 Gln Ala Gln Arg Ser Gly Asn LeuLys Lys Ala Pro Leu Lys Leu 530 535 540 Gly Asn Lys Ala Tyr Pro Ala GlyGlu Ala Ala Pro Gly Thr Tyr 545 550 555 Val His Glu Arg 2 1680 DNAPseudomonas jessenii P161 ; BP-7376 2 atgagtaaca agaataacga tgacttgaagagtcaagcct cggaaaacac 50 cttggggctg aatcctgtcg ttggactgcg tggaaaggatctactggctt 100 ctgctcgaat ggtgctcagg caggccatca agcaaccgat tcacagcgcc150 aggcatgtcg ctcatttcgg cctggaactc aagaacgtgc tgctcggcaa 200atccgagctg ctaccgacca gcgatgaccg tcgtttcgcg gatccggcct 250 ggagccagaacccgctctac aaacgttatc tgcaaaccta cctggcgtgg 300 cgcaaggaac tccacgactggatcgacgac agcaacctgc cggccaagga 350 cgtcagccgc gggcacttcg tgatcaacctcatgaccgag gccttcgccc 400 cgaccaacac ggcggccaac ccggcggcgg tcaaacgcttcttcgaaacc 450 ggtggcaaga gcctgctcga tggcctctcg catctggcca aggacctggt500 acataacggc ggcatgccga gccaggtcaa catgggcgca ttcgaggtcg 550gcaagaccct tggcgtgacc gagggcgcgg tggtctttcg caatgacgtg 600 ctggaactgatccagtacaa accgatcacc gagcaggtgc atgaacgccc 650 actgctggtg gtaccgccacagatcaacaa gttctacgtt ttcgacctga 700 gcccggaaaa gagcctggcg cgattctgcctgcgcaacaa cgtgcagacc 750 ttcatcgtca gctggcgcaa cccgaccaag gagcagcgcgagtggggcct 800 gtcgacctac atcgaagcgc tcaaggaagc ggttgatgtg gtcaccgcca850 tcaccggcag caaagacgtg aacatgctcg gtgcctgctc cggcggcatc 900acctgcaccg cgctgctggg ccactacgca gcaatcggcg agaacaaggt 950 caacgccctgaccctgctgg tcagcgtgct cgacaccacc ctggacagcg 1000 acgtggccct gttcgtcgacgagcagaccc tcgaagccgc caagcgccag 1050 tcgtaccagg ccggtgtact cgaaggccgtgacatggcga aagtcttcgc 1100 ctggatgcgc cccaacgacc tgatctggaa ctactgggtcaacaactact 1150 tgttgggcaa cgagccgccg gtattcgaca ttctgttctg gaacaacgac1200 accacccggt tgcccgccgc gttccatggc gacctgatcg agatgttcaa 1250aagtaacccg ttgacccgtg ccgatgcact ggaagtgtgc ggtacgccga 1300 tcgatctgaagaaagtcacc gccgacatct tctcgctggc cggcaccagc 1350 gaccacatta ccccgtggcgctcctgctac aagtcggcgc aactgttcgg 1400 cggcaacgtt gaattcgtat tgtccagcagcgggcacatc cagagcattc 1450 tgaacccgcc gggcaatccg aaatcgcgtt acatgaccagcaccgaaatg 1500 cccgccaatg ccgatgactg gcaggaagag tcgaccaagc acgccgactc1550 ctggtggctg cactggcagg catggcaggc acagcgttcg ggcaacctga 1600aaaaagcccc gctgaaattg ggcaacaagg cctatccagc gggtgaagcc 1650 gcaccgggcacttacgtgca tgagcggtaa 1680 3 560 PRT Pseudomonas jessenii P161 ; BP-73763 Met Arg Glu Lys Pro Ala Arg Asp Ser Leu Pro Thr Pro Ala Ala 1 5 10 15Phe Ile Asn Ala Gln Ser Ala Ile Thr Gly Leu Arg Gly Arg Asp 20 25 30 LeuLeu Ser Thr Leu Arg Ser Val Ala Ala His Gly Leu Arg Asn 35 40 45 Pro ValHis Ser Ala Arg His Ala Leu Lys Leu Gly Gly Gln Leu 50 55 60 Gly Arg ValLeu Leu Gly Glu Thr Leu His Pro Thr Asn Pro Gln 65 70 75 Asp Thr Arg PheAla Asp Pro Ala Trp Ser Leu Asn Pro Phe Tyr 80 85 90 Arg Arg Ser Leu GlnAla Tyr Leu Ser Trp Gln Lys Gln Val Lys 95 100 105 Ser Trp Ile Asp GluSer Asn Met Ser Pro Asp Asp Arg Ala Arg 110 115 120 Ala His Phe Ala PheAla Leu Leu Asn Asp Ala Val Ser Pro Ser 125 130 135 Asn Thr Leu Leu AsnPro Leu Ala Val Lys Glu Phe Phe Asn Ser 140 145 150 Gly Gly Asn Ser LeuVal Arg Gly Ile Gly His Leu Val Asp Asp 155 160 165 Leu Leu His Asn AspGly Leu Pro Arg Gln Val Thr Lys Gln Ala 170 175 180 Phe Glu Val Gly LysThr Val Ala Thr Thr Thr Gly Ala Val Val 185 190 195 Phe Arg Asn Glu LeuLeu Glu Leu Ile Gln Tyr Lys Pro Met Ser 200 205 210 Glu Lys Gln Tyr SerLys Pro Leu Leu Val Val Pro Pro Gln Ile 215 220 225 Asn Lys Tyr Tyr IlePhe Asp Leu Ser Pro His Asn Ser Phe Val 230 235 240 Gln Tyr Ala Leu LysAsn Gly Leu Gln Thr Phe Met Ile Ser Trp 245 250 255 Arg Asn Pro Asp ValArg His Arg Glu Trp Gly Leu Ser Thr Tyr 260 265 270 Val Glu Ala Val GluGlu Ala Met Asn Val Cys Arg Ala Ile Thr 275 280 285 Gly Ala Arg Glu ValAsn Leu Met Gly Ala Cys Ala Gly Gly Leu 290 295 300 Thr Ile Ala Ala LeuGln Gly His Leu Gln Ala Lys Arg Gln Leu 305 310 315 Arg Arg Val Ser SerAla Thr Tyr Leu Val Ser Leu Leu Asp Ser 320 325 330 Glu Leu Asp Ser ProAla Ser Leu Phe Ala Asp Glu Gln Thr Leu 335 340 345 Glu Ala Ala Lys ArgArg Ser Tyr Gln Lys Gly Val Leu Asp Gly 350 355 360 Arg Asp Met Ala LysVal Phe Ala Trp Met Arg Pro Asn Asp Leu 365 370 375 Ile Trp Ser Tyr PheVal Asn Asn Tyr Leu Leu Gly Lys Glu Pro 380 385 390 Pro Ala Phe Asp IleLeu Tyr Trp Asn Asn Asp Ser Thr Arg Leu 395 400 405 Pro Ala Ala Leu HisGly Asp Leu Leu Asp Phe Phe Lys His Asn 410 415 420 Pro Leu Thr His ProGly Gly Leu Glu Val Cys Gly Thr Pro Ile 425 430 435 Asp Leu Gln Lys ValThr Val Asp Ser Phe Ser Val Ala Gly Ile 440 445 450 Asn Asp His Ile ThrPro Trp Asp Ala Val Tyr Arg Ser Ala Leu 455 460 465 Leu Leu Gly Gly GluArg Arg Phe Val Leu Ser Asn Ser Gly His 470 475 480 Val Gln Ser Ile LeuAsn Pro Pro Ser Asn Pro Lys Ala Asn Tyr 485 490 495 Val Glu Asn Gly LysLeu Ser Ser Asp Pro Arg Ala Trp Tyr Tyr 500 505 510 Asp Ala Arg His ValAsp Gly Ser Trp Trp Thr Gln Trp Leu Ser 515 520 525 Trp Ile Gln Glu ArgSer Gly Ala Gln Lys Glu Thr His Met Ala 530 535 540 Leu Gly Asn Gln AsnTyr Pro Pro Met Glu Ala Ala Pro Gly Thr 545 550 555 Tyr Val Arg Val Arg560 4 1683 DNA Pseudomonas jessenii P161 ; BP-7376 4 atgcgcgagaaaccagcgag ggattcctta ccgactcccg ccgcgttcat 50 caatgcacag agtgcgattaccggcctgcg cggtcgggat ctgttatcga 100 ccctgcgtag tgtggccgcc catggcttgcgcaatccggt gcacagtgcc 150 cgacatgccc tcaaactcgg cggccagctc ggtcgtgtgttgctgggcga 200 aaccctgcac ccgaccaacc cgcaggacac tcgcttcgcc gatccggcgt250 ggagcctcaa cccgttttat cggcgcagcc tgcaggctta tctgagctgg 300cagaagcagg tcaaaagctg gatcgacgag agcaacatga gcccggacga 350 ccgtgcccgcgcccacttcg ctttcgcctt gctcaacgac gccgtatcgc 400 cctccaacac cctgctcaatccattggcgg tcaaggagtt cttcaattcc 450 gggggtaaca gcctggtgcg tggcatcggccatctggtgg acgatctgct 500 gcacaacgat ggcctgcccc ggcaagtcac caagcaagcgttcgaggtcg 550 gcaagacggt cgccaccacc accggtgccg tggtgtttcg caacgaactg600 ctggagttga tccagtacaa gccgatgagc gaaaagcagt attccaagcc 650cctgttggtg gtgccgccgc aaatcaacaa gtactacatt ttcgacctga 700 gcccccacaacagcttcgtc cagtacgcgc tgaaaaacgg cctgcaaacc 750 ttcatgatca gctggcgcaacccggatgtg cgtcaccgcg aatgggggct 800 ctcgacctac gtggaagccg tggaagaagccatgaatgtc tgccgggcga 850 tcaccggtgc acgcgaggtc aacctgatgg gcgcctgcgccggcgggctg 900 accattgccg cgttgcaggg ccacttgcaa gccaaacggc agctgcgcag950 ggtgtccagt gcaacgtatc tggtgagcct gctcgacagt gaactggaca 1000gccccgcttc actgttcgcc gacgaacaga ctctggaggc tgccaagcgt 1050 cgctcctatcagaaaggtgt gctggacggc cgcgacatgg ccaaggtctt 1100 cgcctggatg cgccccaacgatttgatctg gagctacttc gtcaacaact 1150 acctgttggg caaggagccg ccggcgttcgacatcctcta ctggaacaac 1200 gacagcacgc gcttgcctgc cgccctgcat ggcgacctgctggacttctt 1250 caagcacaac ccgctgaccc acccgggcgg cctggaagtg tgtggcacgc1300 cgatcgattt gcagaaggtc accgttgaca gcttcagcgt cgccggcatc 1350aacgatcaca tcacgccttg ggatgcggtg tatcgctcgg cgctgttgct 1400 cggtggcgagcggcgcttcg tgctgtccaa cagcggccat gtgcagagca 1450 tcctcaaccc gccgagcaacccgaaagcca actacgtcga aaacggcaag 1500 ctgagcagcg acccccgcgc ctggtactacgacgccaggc atgtcgacgg 1550 cagttggtgg acccaatggc tgagctggat tcaggaacgctccggcgcgc 1600 agaaggaaac ccacatggcg ctcggcaacc agaactatcc accgatggaa1650 gctgcgcccg gtacctacgt acgtgtgcgc tga 1683 5 1501 DNA Pseudomonasjessenii P161 ; BP-7376 cDNA to 16S rRNA 5 tgaacgctgg cggcaggcctaacacatgca agtcgagcgg atgacgggag 50 cttgctcctg aattcagcgg cggacgggtgagtaatgcct aggaatctgc 100 ctggtagtgg gggacaacgt ctcgaaaggg acgctaataccgcatacgtc 150 ctacgggaga aagcagggga ccttcgggcc ttgcgctatc agatgagcct200 aggtcggatt agctagttgg tgaggtaatg gctcaccaag gcgacgatcc 250gtaactggtc tgagaggatg atcagtcaca ctggaactga gacacggtcc 300 agactcctacgggaggcagc agtggggaat attggacaat gggcgaaagc 350 ctgatccagc catgccgcgtgtgtgaagaa ggtcttcgga ttgtaaagca 400 ctttaagttg ggaggaaggg cattaacctaatacgttagt gttttgacgt 450 taccgacaga ataagcaccg gctaactctg tgccagcagccgcggtaata 500 cagagggtgc aagcgttaat cggaattact gggcgtaaag cgcgcgtagg550 tggtttgtta agttggatgt gaaagccccg ggctcaacct gggaactgca 600ttcaaaactg acaagctaga gtatggtaga gggtggtgga atttcctgtg 650 tagcggtgaaatgcgtagat ataggaagga acaccagtgg cgaaggcgac 700 cacctggact gatactgacactgaggtgcg aaagcgtggg gagcaaacag 750 gattagatac cctggtagtc cacgccgtaaacgatgtcaa ctagccgttg 800 ggagccttga gctcttagtg gcgcagctaa cgcattaagttgaccgcctg 850 gggagtacgg ccgcaaggtt aaaactcaaa tgaattgacg ggggcccgca900 caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc 950aggccttgac atccaatgaa ctttccagag atggatgggt gccttcggga 1000 acattgagacaggtgctgca tggctgtcgt cagctcgtgt cgtgagatgt 1050 tgggttaagt cccgtaacgagcgcaaccct tgtccttagt taccagcacg 1100 taatggtggg cactctaagg agactgccggtgacaaaccg gaggaaggtg 1150 gggatgacgt caagtcatca tggcccttac ggcctgggctacacacgtgc 1200 tacaatggtc ggtacagagg gttgccaagc cgcgaggtgg agctaatccc1250 acaaaaccga tcgtagtccg gatcgcagtc tgcaactcga ctgcgtgaag 1300tcggaatcgc tagtaatcgc gaatcagaat gtcgcggtga atacgttccc 1350 gggccttgtacacaccgccc gtcacaccat gggagtgggt tgcaccagaa 1400 gtagctagtc taaccttcgggaggacggtt accacggtgt gattcatgac 1450 tggggtgaag tcgtaccaag gtagccgtaggggaacctgc ggctggatca 1500 c 1501 6 20 DNA Artificial Sequence Primerfor PCR multiplication 6 tgctggaact gatccagtac 20 7 23 DNA ArtificialSequence Primer for PCR multiplication 7 gggttgagga tgctctggat gtg 23 830 DNA Artificial Sequence Primer for PCR multiplication 8 ctacaaagcttgacccggta ctcgtctcag 30 9 29 DNA Artificial Sequence Primer for PCRmultiplication 9 gcgagcaagc ttgctcctac agggatagc 29 10 29 DNA ArtificialSequence Primer for PCR multiplication 10 gttttaagct tgaagacgaaggagtgttg 29 11 30 DNA Artificial Sequence Primer for PCR multiplication11 ctcctacaag cttggagact gactgtggcc 30

What is claimed is:
 1. An isolated polyhydoxyalkanoate synthase havingan amino acid sequence of SEQ ID NO.
 1. 2. An isolatedpolyhydroxyalkanoate synthase having a modified amino acid sequencecomprising an amino acid sequence of SEQ ID NO. 1 in which at least oneamino acid is deleted, substituted or added, wherein said modified aminoacid sequence hybridizes to the amino acid sequence of said SEQ ID NO. 1under stringent condictions.